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EP3359171B1 - Utilisation d'akkermansia muciniphila pour traiter des états inflammatoires - Google Patents

Utilisation d'akkermansia muciniphila pour traiter des états inflammatoires Download PDF

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Publication number
EP3359171B1
EP3359171B1 EP16782086.9A EP16782086A EP3359171B1 EP 3359171 B1 EP3359171 B1 EP 3359171B1 EP 16782086 A EP16782086 A EP 16782086A EP 3359171 B1 EP3359171 B1 EP 3359171B1
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composition
akkermansia muciniphila
strains
akkermansia
prophylaxis
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EP3359171A1 (fr
EP3359171C0 (fr
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Liam O'mahony
Cezmi A AKDIS
David Michalovich
Edith M HESSEL
James R. Brown
David N. MAYHEW
Sorif UDDIN
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Schweizerisches Forschungsinstitut fur Hochgebirgsklima und Medizin In Davos
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Schweizerisches Forschungsinstitut fur Hochgebirgsklima und Medizin In Davos
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • the invention relates to the use of Akkermansia muciniphila as an immunomodulatory biotherapeutic agent for treating asthma. More generally, it relates to use of Akkermansia muciniphila, a mucin-degrading bacterial species found in the human gut, for treating undesirable inflammatory activity unrelated to any metabolic disorder and /or obesity. In this context, "unrelated to” will be understood to mean “not caused by”. Of especial interest is use of Akkermansia muciniphilia in the prophylaxis and/or treatment of airway inflammation in asthmatic patients identified as having a lower level of that microbe in the gut compared to healthy individuals. Commonly such asthmatics can be expected to have asthma which can be categorized as severe.
  • the human microbiome is a microbial community, which can be described as the sum of all microbial life living in or on specific sites of the human body.
  • Recent advances in DNA sequencing techniques have facilitated more in-depth analysis of the microbiomes of the gut, skin, genito-urinary tract and the lung, revealing a microbial super organ residing symbiotically with host mucosal surfaces. It is becoming better appreciated that the composition and activity of the microbiome has significant metabolic, nutritional and immunological effects on the host (1).
  • the microbiome evolves within a host from birth, constantly being fine-tuned to maintain a homeostatic balance with the host's immune system. This evolution is influenced by host factors, such as the adaptive and innate immune responses, external factors such as diet, medication and toxin exposure, and illness.
  • the gastrointestinal tract has the greatest number and diversity of microbes, with approximately 100 trillion microbes residing in the gut (2 to 10 times the total number of human cells in the entire body), while the collective genomes of the gut microbiome contain approximately 100 times more genes than the human genome. These microbes are highly adapted to survive within complex community structures, requiring nutrients from other microbes and/or host processes. Interestingly, using sequencing approaches, over 1,000 different bacterial species have been identified within the gut microbiome. However, a specific individual's microbiome typically contains only 300-500 different species, leading to enormous inter-individual variability in microbiome composition.
  • microbiome supports the development of epithelial barrier function and integrity, while promoting potent tolerance and protective immune mechanisms within mucosal tissues (4).
  • Microbes have direct effects on host immune responses and metabolites derived from microbial fermentation of nutrients within the gut not only contribute to host energy intake, but also significantly influence host immunological responses (e.g. short-chain fatty acids and histamine) (5, 6).
  • Appropriate cellular and molecular networks involve innate pattern recognition receptor activation, T and B cell polarization and expansion, secretion of a wide range of effector and regulatory cytokines and host metabolites.
  • this trialogue between the microbiome, immune cells and tissue cells within the gut results in the establishment of optimal digestive capabilities, gut motility, immune tolerance to foods and certain microbial antigens, and protection against pathogens.
  • Akkermansia species are commensal microorganisms. They have been isolated from the microbial flora within the human gastrointestinal tract (11). The immune system within the gastrointestinal tract cannot have a pronounced reaction to members of this flora, as the resulting inflammatory activity would also destroy host cells and tissue function. Therefore, some mechanism(s) exist whereby the immune system can recognize commensal non-pathogenic members of the gastrointestinal flora as being different to pathogenic organisms.
  • Akkermansia muciniphila has been previously reported to be reduced in faecal samples from obese individuals and patients with inflammatory bowel disease (13). Akkermansia muciniphila is associated with a healthier metabolic status and better clinical outcomes after a calorie restriction intervention in overweight/obese adults (14). Indeed, published International Patent Application WO 2014/075745 , and published International Application WO 2014/076246 claiming priority therefrom, concern the protective effects of Akkermansia muciniphila in obese individuals and propose administration of Akkermansia muciniphila to individuals exhibiting obesity and / or metabolic disorders such as type 2 diabetes to provide various beneficial effects (15).
  • Severe asthma as referred to herein can be understood and defined as follows: When a diagnosis of asthma is confirmed and comorbidities have been addressed, severe asthma is defined as "asthma which requires treatment with high dose inhaled corticosteroids (ICS) plus a second controller (and/or systemic CS) to prevent it from becoming “uncontrolled” or which remains “uncontrolled” despite this therapy.” (18)
  • ICS inhaled corticosteroids
  • CS systemic CS
  • the present invention thus provides a composition comprising one or more Akkermansia muciniphila strains for use in the prophylaxis and/or treatment of airway inflammation of asthma.
  • Such a composition may be a probiotic composition for delivery of the one or more Akkermansia muciniphilia strains in the GI tract, preferably for oral administration, e.g. in the form of a beverage or other form of nutritional composition for oral consumption. It may be in the form of a pharmaceutical composition for administration of one of more Akkermansia muciniphila strains in the GI tract, preferably by oral administration.
  • the composition may be a strain-release composition which is not ingested in which case said use further comprises the pre-step of contacting said composition with a liquid whereby the one or more probiotic strains are released into the liquid and the resulting probiotic liquid composition is then ingested.
  • the probiotic composition employed may alternatively be formulated and packaged for aerosol delivery.
  • Akkermansia muciniphila strains for use in applying the invention will be understood to include naturally-occurring strains and additionally variants and genetically-modified mutants of naturally-occurring Akkermansia muciniphila strains, in any form, provided that desired immunomodulatory effect when in the gut is maintained.
  • the present invention provides a composition comprising one or more Akkermansia muciniphila strains for use in the prophylaxis and/or treatment of undesirable inflammatory activity wherein said inflammatory activity is not caused by a metabolic disorder and/or obesity and is remote from the gastrointestinal tract. More particularly, the undesirable inflammatory activity of concern is airway inflammatory activity of asthma
  • the invention thus provides a composition comprising one or more Akkermansia muciniphila strains for use in the prophylaxis and/or treatment of airway inflammation of asthma, especially in asthma patients with a lower than normal level of Akkermansia muciniphilia in the gut.
  • Akkermansia muciniphila strains for use in the prophylaxis and/or treatment of airway inflammation of asthma, especially in asthma patients with a lower than normal level of Akkermansia muciniphilia in the gut.
  • Akkermansia muciniphilia are severe asthmatics.
  • Asthma patients with a lower than normal gut level of Akkermansia muciniphila may be conveniently pre-identified by application of various quantitative diagnostic tests to faecal samples as further discussed below.
  • the one or more Akkermansia muciniphila strains employed may be naturally-occurring strains derived from human faeces or biopsies, or naturally-occurring variants thereof, which have the desired immunomodulatory effect in the human gut. Genetically modified mutants of naturally-occurring Akkermansia muciniphila strains may also be employed provided they retain the desired activity. This may for example be equated with ability to induce ILT4 expression in human dendritic cells in vitro. A suitable assay for this purpose is set out in the exemplification below.
  • a composition for use in accordance with the invention may additionally include one or more probiotic strains of another bacterial species such as a Lactobacillus or Bifidobacterium, for example one or more Akkermansia muciniphila strains may be combined with Bifidobacterium longum infantis 35624 or a variant or mutant thereof as described in EP1141235 .
  • probiotic strains of another bacterial species such as a Lactobacillus or Bifidobacterium
  • Akkermansia muciniphila strains may be combined with Bifidobacterium longum infantis 35624 or a variant or mutant thereof as described in EP1141235 .
  • any other probiotic bacterial strain with immunological benefit might be considered.
  • the cells of a different species may be administered sequentially or simultaneously in a different composition.
  • a composition for use in accordance with the invention may be for direct delivery of one more Akkermansia muciniphila strains into the GI tract.
  • a composition for use in accordance with the invention will be a probiotic composition suitable for direct oral consumption.
  • a strain-release composition may be provided, which is not ingested but is contacted with a liquid whereby said one or more strains are released into said liquid and the resulting probiotic liquid composition is then consumed.
  • an oil suspension within a drinking straw whereby the one or more strains are transferred to a liquid drawn through the straw, e.g. a juice or milk from a drink carton.
  • a liquid drawn through the straw e.g. a juice or milk from a drink carton.
  • Such straws housing probiotic strains are described, for example, in European Patent no.1224128 (Biogaia AB ).
  • a probiotic composition for oral administration in accordance with the invention may include one or more Akkermansia muciniphila strains and an ingestible carrier. It may preferably be in the form of a beverage, e.g. drinking yoghurt, juice or milk, or other form of nutritional composition. It may be suitable for human and/or animal consumption.
  • the composition may be in the form of a food product such as a yoghurt, cheese, confectionary, cereal or nutritional snack bar. It may be in the form of a nutritional supplement.
  • the one or more probiotic strains may be provided in a nutritional oil suspension, e.g. within a capsule, which will release the probiotic strains in the GI tract.
  • a composition for use in accordance with the invention may be formulated as a pharmaceutical composition including a pharmaceutically acceptable carrier, preferably such a formulation for oral administration including an ingestible carrier.
  • a pharmaceutically acceptable carrier preferably such a formulation for oral administration including an ingestible carrier.
  • Such a composition may be in a solid form such as a tablet, pill, powder, granules, troches or suppository. It may be a controlled-release formulation for release of the one or more probiotic strains in the GI tract. It may be in a liquid form, such as a liposomal composition or suspension, e.g. an oil suspension.
  • Such a liquid composition may be provided within an encapsulating substance to provide a capsule or microcapsules, again for release of the one or more probiotic strains in the GI tract. It may be a formulation for aerosol delivery.
  • a pharmaceutical composition for use in accordance with the invention may additionally include a non-microbial therapeutic agent, e.g. a chemical drug entity or a therapeutic biologic, suitable for the prophylaxis and/or treatment of the undesired inflammatory activity of concern or an associated condition.
  • a non-microbial therapeutic agent e.g. a chemical drug entity or a therapeutic biologic, suitable for the prophylaxis and/or treatment of the undesired inflammatory activity of concern or an associated condition.
  • a composition for use in accordance with the invention will be formulated to provide an appropriate unit dosage of colony-forming units (cfu). This will vary with the form of administration and may be varied, for example, depending on whether the individual to be treated is a child or adult. For example a dosage of at least about 10 4 cfu, more preferably at least about 10 5 cfu or at least about 10 6 cfu, e.g. about 10 6 - 10 10 cfu may be contemplated at least once per day. Administration will desirably be continued so that the level of Akkermansia muciniphila in the GI tract is at least restored to or maintained at around the normal level to be expected for a healthy individual.
  • cfu colony-forming units
  • Akkermansia muciniphila e.g. Akkermansia muciniphila Derrien et al. (ATTC ® BAA-835 TM ) isolated from human faeces and obtainable from the ATCC as a freeze dried preparation, or Akkermansia muciniphilia similarly isolated from human faeces or biopsies, may be grown anaerobically in a mucin-based medium as previously described by Derrien et al. (11) and subsequently incorporated into an appropriate composition for administration as discussed above.
  • Akkermansia muciniphila thus cultured may be washed and at least initially suspended in anaerobic phosphate buffered saline (PBS), including 25% (v/v) glycerol, e.g. to an end concentration of 10 10 cfu /ml under strict anaerobic conditions.
  • PBS phosphate buffered saline
  • Storage may be at -80° C with subsequent thawing and possible further dilution, e.g. with anaerobic PBS, as previously described (15, 16).
  • Storage prior to use in accordance with the invention will be such as to protect from contact with air. Storage may be for example within lipid gels or drops.
  • compositions for use in accordance with the invention will generally be stored prior to use in a sealed container or packaging to aid avoidance of prolonged contact with air. Storage may be for example in the form of a digestible solid composition, e g. pills, provided in sealed compartments of a blister pack.
  • a composition for use in accordance with the invention by delivery to the GI tract may additionally be supplemented with one or more prebiotic substances.
  • prebiotic substances are recognized to promote growth of one or more probiotic strains in the GI tract, but need not be metabolized by a human.
  • the term 'prebiotic' will be understood to refer to any non-viable food component that can be specifically fermented in the colon by indigenous bacteria and which is considered of positive value.
  • Types of prebiotics of interest include non-digestible oligosaccharides including those containing one or more of fructose, xylose, galactose, glucose and mannose. Soya may be employed as a prebiotic.
  • the combined administration of a bacterial strain with one or more prebiotic substances may enhance the growth of the strain in vivo resulting in a more pronounced health benefit, and is termed synbiotic.
  • a prebiotic substance which will promote growth in the GI tract of Akkermansia muciniphila.
  • oligofructose will promote growth of Akkermansia muciniphila in the GI tract of mice (16).
  • oligofructose e.g. a commercially available oligofructose preparation such as an Orafti ® oligofructose preparation, e.g. Orafti ® P95 as supplied by Beneo GmbH
  • a prebiotic may be administered in the same composition or separately, either simultaneously or sequentially.
  • compositions containing Akkermansia muciniphilia are employed as described above in combination with a diagnostic test to determine Akkermansia muciniphila abundance in an individual with an aim of treating identified Akkermansia muciniphilia deficiency.
  • a diagnostic test may take the form of faecal bacterial microbiome 16S rRNA gene sequencing, PCR specific detection of Akkermansia muciniphilia DNA or application of other nucleic acid hybridisation or sequencing technologies, or specific culture on selective media, for specific detection of Akkermansia muciniphilia.
  • the 75% percentile for relative abundance of Akkermansia muciniphila in severe asthma patients is 0.01422, which means that 1.422% of identifiable sequences are Akkermansia muciniphila. This suggests that Akkermansia muciniphila treatment may be most effective in asthma patients whom have a relative abundance of less than about 1.4%.
  • the 75% percentile for Akkermansia muciniphila in severe asthma patients is 60.1 copy number/ng DNA, suggesting that Akkermansia muciniphila treatment may be most effective in asthma patients whom have a detected copy number of less than about 60 per ng of total bacterial DNA.
  • Akkermansia muciniphila levels are significantly reduced in faecal samples from asthma patients displaying partly controlled or uncontrolled asthma symptoms. Furthermore, consumption of Akkermansia muciniphila has been found to significantly reduce recruitment of disease-causing cells to the lungs in four different murine models of respiratory inflammation. In addition, Akkermansia muciniphila has immunomodulatory effects; it can modulate cytokine secretion by human dendritic cells in vitro. Indeed, stimulation of the inhibitory molecule LILRB2 (ILT4) by Akkermansia muciniphila is surprisingly significantly higher than that observed for other commensal microbes typically used as probiotics.
  • LILRB2 inhibitory molecule
  • Neutrophils are a type of phagocyte and are normally found in the bloodstream.
  • neutrophils are one of the first-responders of inflammatory cells to migrate towards the site of inflammation. They migrate through the blood vessels, then through interstitial tissue, following chemical signals in a process called chemotaxis. They are the predominant cells in pus, accounting for its whitish/yellowish appearance.
  • Neutrophils release a wide range of proteins, which help to destroy a pathogen, but also have detrimental effects on host cells at higher concentrations. Asthma and asthma exacerbations can be associated with high neutrophil or high eosinophil numbers within the lung, both of which contribute to the disease.
  • Eosinophils are white blood cells and one of the immune system components responsible for combating parasites and certain infections. Along with mast cells, they also control mechanisms associated with allergy and asthma. The presence of eosinophils in the lung or skin is associated with disease. Eosinophils persist in the circulation for 8-12 hours, and can survive in tissue for an additional 8-12 days in the absence of stimulation. Eosinophils are important mediators of allergic responses and asthma pathogenesis and are associated with disease severity. Following activation, eosinophils release a range of powerful molecules including cationic granule proteins, reactive oxygen species, lipid mediators, enzymes, growth factors and cytokines. Many of the mediators released by eosinophils are toxic at high levels to host cells.
  • Assays described below measured IgE, ILT4, IL-4, IL5, IL-10 and Foxp3 + regulatory T cells.
  • IL-4 is a 15-kd monomer (129 amino acids) produced by Th2 cells, basophils, mast cells, and eosinophils.
  • IL-4 receptors There are two types of IL-4 receptors. Type I IL-4R binds only IL-4 and consists of 2 receptor chains: IL-4R ⁇ (CD124) and the common ⁇ c (CD132). Type II IL-4R binds IL-4 and IL-13 and consists of the IL-4R ⁇ and the IL-13R ⁇ 1 chains.
  • a pleiotropic cytokine, IL-4 promotes allergic conditions and the protective immune response against helminths and other extracellular parasites.
  • IL-4 is the major stimulus of Th2-cell development; it also suppresses Th1-cell development and induces IgE class-switching in B cells.
  • IL-4 increases the expression of class II MHC molecules on B cells, upregulates B-cell receptors, increases expression of CD23, prolongs lifespans of T and B cells in culture, and mediates tissue adhesion and inflammation.
  • IL-4 and IL-4R ⁇ knockout mice have defects in Th2-cell differentiation and reduced serum levels of IgG1 and IgE.
  • IL-5 is mainly produced by CD4+ Th2 cells, activated eosinophils, mast cells, CD8+Tc2 cells, ⁇ T cells, NK cells, NKT cells, and CD4-ckit-CD3 ⁇ -IL-2R ⁇ + cells in Peyer patches. Its receptor shares the ⁇ -chain (CD131) with IL-3 and GM-CSF. IL-5 promotes proliferation, activation, differentiation, survival, and adhesion of eosinophils. Th2 cells that secrete IL-5 recruit eosinophils and contribute to the induction of airway hyper-reactivity in patients with asthma.
  • IL-5-deficient mice develop normally but are resistant to induction of experimental asthma, display reduced expulsion of Nippostrongylus brasiliensis, and have fewer IgA+ antibody cells in the lamina propria compared with control mice.
  • Clinical trials targeting IL-5 have produced mixed results, but patients with refractory eosinophilic asthma were reported to have reduced numbers of exacerbations and eosinophils in sputum and blood and increased quality of life (21).
  • FOXP3 (forkhead box P3) also known as scurfin is a protein involved in immune system responses.
  • a member of the FOX protein family, FOXP3 is a master regulator (transcription factor) in the development and function of regulatory T cells.
  • Regulatory T cells prevent excessive immune responses, which could damage host tissues.
  • autoimmune disease a deficiency of regulatory T cell activity can allow other autoimmune cells to attack the body's own tissues. Defects in regulatory T cell induction and/or activity have been demonstrated in multiple allergy and asthma models.
  • Immunoglobulin-like transcript 4 can be expressed on dendritic cells and is bound by HLA-G.
  • the engagement of ILT4 by HLA-G tetramers prevents the up-regulation of costimulatory molecule expression, inhibits dendritic cell maturation and promotes the differentiation of anergic/suppressor CD4 + T cells.
  • differentiation of the critical immunoregulatory IL-10 secreting T cell subset, Tr1 cells is dependent on the IL-10-dependent ILT4/HLA-G pathway (22). These Tr1 cells play a central role in tolerance induction and contribute to the suppression of aberrant inflammatory activity, such as that observed in the inflamed lung of asthma patients.
  • IL-10 is an anti-inflammatory cytokine that is an important regulator of several aspects of immune responses (23).
  • the IL-10 gene maps to a cytokine cluster that includes the genes IL-19, IL-20, IL-24, and IL-26 on chromosome 1q31-32.
  • IL-10 is produced mainly by monocytes, T cells (mainly Tr1 cells), B cells, NK cells, macrophages, and dendritic cells.
  • IL-10 is secreted as a homodimer that consists of 2 subunits, each of 178 amino acids with a molecular weight of approximately 18 kd.
  • IL-10 directly affects antigen-presenting cell functions by down-regulating the expression of MHC class II and costimulatory molecules on the surface of macrophages and monocytes.
  • IL-10 inhibits the expression of many proinflammatory cytokines, chemokines, and chemokine receptors and mediates allergen tolerance in allergen-specific immunotherapy and after exposure to high doses of allergen.
  • IL-10 directly affects T-cell activation by suppressing CD28, CD2, and signaling of the inducible T-cell costimulator via the tyrosine phosphatase SHP-1.
  • IL-10 promotes survival, proliferation, and differentiation of human B cells and increases the production of IgG4.
  • Several mouse models demonstrate the importance of IL-10 in regulation of the inflammatory response. IL-10 knockout mice develop normal lymphocyte and antibody responses but have reduced growth, are anemic, and spontaneously develop chronic colitis.
  • IgE Immunoglobulin E
  • Presence of elevated Immunoglobulin E (IgE) levels in serum is regarded as an integral part of the inflammatory cascade observed in allergic diseases such as asthma.
  • Induction of allergen-specific IgE can be detected in animal models of allergic pulmonary inflammation as well as in human asthmatics and is an indication of the initiation of the relevant cellular (T and B cell axis) and humoral mechanisms that drive development of allergy.
  • modulation of the allergen-specific IgE response by therapeutic intervention points to modulation of the mechanisms that fundamentally underpin development of allergy.
  • the importance of elevated levels of IgE is demonstrated by the fact that anti-IgE antibody therapies have been shown to be important in reducing severe asthmatic exacerbations (24).
  • Example 1 Abundance of bacterial species in faeces from asthma patients.
  • Freshly voided faecal samples were obtained from asthma patients and were stored at -80C until analysis. Upon thawing, DNA was extracted from the faecal samples and the 16s rRNA V4 region was amplified by PCR. These PCR products were sequenced using MiSeq and identical sequences were grouped together into operational taxonomic units (OTUs). OTUs were aligned to known 16s sequences and the relative abundance for each OTU was calculated.
  • OTUs operational taxonomic units
  • a “No” answer to all four questions means that the asthma patient should be considered as well controlled.
  • a “Yes” answer to one or two questions means that the patient is partly controlled, while a “Yes” answer to three or four questions means that the patient has uncontrolled asthma symptoms.
  • HDM allergens are one of the most common aeroallergens that asthma patients are allergic to.
  • Intranasal (i.n.) administration of HDM extract to mice results in lung inflammatory responses.
  • HDM extract i.n. on day 0 (1 ⁇ g), followed by higher dose i.n. administrations on days 7, 8, 9, 10 and 11 (10 ⁇ g each day).
  • This model is considered to be an acute challenge model. Animals were euthanized on day 14 and bronchoalveolar lavages (BAL) were obtained for enumeration of inflammatory cells.
  • BAL bronchoalveolar lavages
  • Akkermansia muciniphila (from a stock culture of the inventors) was administered daily (approximately 1 ⁇ 10 8 cells) by oral gavage, beginning at day -5 until the end of the study. Akkermansia muciniphila was grown for 16 hours in 10 mL aliquots of anaerobic Mucin v3 media (10% inoculation) at 37 °C. Nitrogen (and boiling) was used to remove the presence of oxygen from Mucin v3 media.
  • Mucin v3 media contains peptone, yeast extract, KH 2 PO 4 , NaCl, (NH 4 )SO 4 , MgSO 4 , CaCl 2 , NaHCO 3 , glucose, mucin type II, hemin, cysteine and water.
  • Negative control animals received NaCl i.n. on the same days as the positive control animals received HDM extracts i.n.
  • HDM extract is composed of many different substances that include proteolytic enzymes (Derp) and LPS and can therefore cause inflammatory responses that involve a vast array of interactions between structural and immune cells (lymphocytic and myeloid) and mediators (26-28).
  • mice Female BALB/c mice (8-10 weeks of age) were intra-nasally challenged with either sterile saline or 25 ⁇ g of HDM extract in a volume of 50 ⁇ l (Greer labs, US, batch number: 218862) 5 days a week, for 3 weeks (sensitization period, days 1-19).
  • This model is different to that described above and is considered to be a chronic model of respiratory inflammation.
  • mice were intra-nasally challenged with either saline or re-challenged with HDM (100 ⁇ g) in a total volume of 50 ⁇ l.
  • HDM re-challenge in this model has been shown to result in granulocytic and lymphocytic infiltration into the lung over a 7 day period (days 34 to 40).
  • mice were dosed by oral gavage (0.2ml) with either media control or Akkermansia muciniphila once a day, starting on day 20 (post cessation of HDM sensitization). Oral dosing continued throughout the resolution period and post HDM re-challenge and finally ceasing on day 39.
  • mice were sacrificed at pre-determined time-points during the resolution phase of the experiment (day 28) and post HDM re-challenge (4hrs (day 34), 24hrs (day 35) and 168hrs (day 40).
  • Cells present in the broncho-alveolar lavage fluid (BALF) and lung tissue were analyzed and quantified using multi-colour flow cytometry.
  • BALF broncho-alveolar lavage fluid
  • Akkermansia muciniphila feeding resulted in enhanced clearance of inflammatory cells (granulocytic and lymphocytic) present in the BALF during the resolution phase of the experiment ( Fig. 4A and 4B ).
  • Infiltration of eosinophils into the BALF after HDM re-challenge was also significantly suppressed by Akkermansia muciniphila feeding ( Fig. 5A ).
  • Numbers of infiltrating inflammatory eosinophils (Siglec-F high eos) were also significantly inhibited in the lung tissue of mice fed with Akkermansia ( Fig. 5B and C ).
  • Akkermansia muciniphila was also able to significantly inhibit the induction of HDM-specific IgE in the serum at day 40 (168hrs post HDM re-challenge ( Fig. 6 )).
  • the HDM mouse model examines the response to an allergenic extract; the inventors also wished to test the influence of Akkermansia muciniphila in an eosinophil dominant model of lung disease, which is driven by a single allergen.
  • Mice were sensitized to the protein ovalbumin (OVA) by intraperitoneal injection (with the adjuvant alum) on days 0, 14 and 21 followed by repeated OVA aerosol challenge on days 26-28. Animals were euthanized on day 29 for analysis of lung disease parameters.
  • OVA ovalbumin
  • Akkermansia muciniphila purchased from the ATCC [Akkermansia muciniphila Derrien et al.
  • Isolated cells from excised lung tissue were also re-stimulated with OVA in vitro to assess the cytokine response to OVA.
  • Lymphocytes from OVA allergic animals secreted significantly more IL-4 and IL-5 in response to OVA compared to lymphocytes from non-allergic animals ( Fig. 8A and 8B ), as expected.
  • this increase in IL-4 and IL-5 secretion was not observed with lung tissue cells from animals treated with Akkermansia muciniphila by oral gavage.
  • Dendritic cells are one of the important cell types within the gut mucosa, which sample luminal bacteria and induce potent polarizing effects on downstream lymphocyte responses (29).
  • Akkermansia muciniphila stimulated dendritic cells to express high levels of immune regulatory molecules, which would promote the downstream anti-inflammatory effects observed in the lung.
  • MDDCs Human monocyte-derived dendritic cells
  • GM-CSF Human monocyte-derived dendritic cells
  • IL-4 Human monocyte-derived dendritic cells
  • Akkermansia muciniphila was prepared by growing for 16 hours in 10 mL aliquots of anaerobic Mucin v3 media (10% inoculation) at 37°C. Bacterial cell number was determined by microscopy and bacterial cells were added to MDDCs at a bacterial cell:MDDC cell ratio of 50:1.
  • Fig. 10 Akkermansia muciniphila induced ILT4 expression on a very large number of MDDCs ( Fig. 10 ). Surprisingly, Akkermansia muciniphila induced ILT4 expression was significantly higher than the effect observed with other probiotic microbes (such as Bifidobacteria or Lactobacilli) or for pathogens such as Staphylococcus aureus ( Fig. 10 ). ILT4 is largely responsible for inducing regulatory T cells, which can secrete IL-10. Increased numbers of IL-10 positive regulatory T cells described in the murine model above may be due to induction of ILT4 expression by dendritic cells.
  • probiotic microbes such as Bifidobacteria or Lactobacilli
  • pathogens such as Staphylococcus aureus
  • Akkermansia muciniphila In addition to inducing very high expression of the regulatory cell surface molecule ILT4, Akkermansia muciniphila also induced very high levels of IL-10 secretion, another anti-inflammatory molecule ( Fig. 11 ).
  • Akkermansia muciniphila was prepared by growing for 16 hours in 10 mL aliquots of anaerobic Mucin v3 media (10% inoculation) at 37°C. Bacterial cell number was determined by microscopy and bacterial cells were added to MDDCs at a bacterial cell:MDDC cell ratio of 10:1, 25:1 or 50:1.

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  1. Composition comprenant une ou plusieurs souches d'Akkermansia muciniphila destinée à être utilisée dans la prophylaxie et/ou le traitement d'une inflammation des voies respiratoires due à l'asthme.
  2. Composition comprenant une ou plusieurs souches d'Akkermansia muciniphila destinée à être utilisée selon la revendication 1, ladite composition étant administrée à un sujet identifié comme ayant un niveau d'Akkermansia muciniphila dans l'intestin inférieur à la normale.
  3. Composition comprenant une ou plusieurs souches d'Akkermansia muciniphila destinée à être utilisée selon la revendication 2, ladite composition étant administrée à un sujet présentant un asthme sévère.
  4. Composition comprenant une ou plusieurs souches d'Akkermansia muciniphila destinée à être utilisée selon l'une quelconque des revendications précédentes, lesdites une ou plusieurs souches étant choisies parmi des souches d'origine naturelle dérivées de biopsies ou de fèces humaines, ou des variantes d'origine naturelle de celles-ci, qui ont un effet immunomodulateur dans l'intestin humain.
  5. Composition comprenant une ou plusieurs souches d'Akkermansia muciniphila destinée à être utilisée selon l'une quelconque des revendications 1 à 3, au moins une telle souche étant un mutant génétiquement modifié d'une souche d'Akkermansia muciniphila d'origine naturelle qui conserve un effet immunomodulateur dans l'intestin humain.
  6. Composition comprenant une ou plusieurs souches d'Akkermansia muciniphila destinée à être utilisée dans la prophylaxie et/ou le traitement d'une activité inflammatoire indésirable selon l'une quelconque des revendications 1 à 5, ladite composition comprenant en outre une ou plusieurs souches probiotiques d'une autre espèce bactérienne telle qu'un Lactobacillus ou un Bifidobacterium, ou lesdites une ou plusieurs souches probiotiques d'une espèce bactérienne autre qu'Akkermansia muciniphila étant administrées soit simultanément soit séquentiellement dans une composition différente.
  7. Composition comprenant une ou plusieurs souches d'Akkermansia muciniphila destinée à être utilisée dans la prophylaxie et/ou le traitement d'une inflammation des voies respiratoires selon l'une quelconque des revendications 1 à 6, ladite composition étant une composition probiotique appropriée pour une administration orale.
  8. Composition comprenant une ou plusieurs souches d'Akkermansia muciniphila destinée à être utilisée dans la prophylaxie et/ou le traitement d'une inflammation des voies respiratoires selon la revendication 7, ladite composition étant une boisson ou une autre forme de composition nutritionnelle.
  9. Composition comprenant une ou plusieurs souches d'Akkermansia muciniphila destinée à être utilisée dans la prophylaxie et/ou le traitement d'une inflammation des voies respiratoires selon l'une quelconque des revendications 1 à 6, ladite composition étant une composition pharmaceutique servant à amener ladite ou lesdites souches dans le tractus gastro-intestinal.
  10. Composition comprenant une ou plusieurs souches d'Akkermansia muciniphila destinée à être utilisée dans la prophylaxie et/ou le traitement d'une inflammation des voies respiratoires selon l'une quelconque des revendications 1 à 6, ladite composition étant préparée et conditionnée pour une administration par aérosol.
  11. Composition comprenant une ou plusieurs souches d'Akkermansia muciniphila destinée à être utilisée dans la prophylaxie et/ou le traitement d'une inflammation des voies respiratoires selon l'une quelconque des revendications 1 à 10, ladite composition comprenant en outre une substance prébiotique, de préférence une substance prébiotique destinée à favoriser la croissance d'Akkermansia muciniphila dans le tractus gastro-intestinal, ou ladite substance prébiotique étant administrée séparément de ladite composition, soit simultanément soit séquentiellement.
  12. Composition comprenant une ou plusieurs souches d'Akkermansia muciniphila destinée à être utilisée dans la prophylaxie et/ou le traitement de l'inflammation des voies respiratoires selon l'une quelconque des revendications 1 à 6, ladite composition étant une composition à libération de souches qui n'est pas ingérée et ladite utilisation comprenant en outre l'étape préalable consistant à mettre en contact ladite composition avec un liquide, moyennant quoi ladite ou lesdites souches sont libérées dans ledit liquide et la composition liquide probiotique ainsi obtenue est ingérée.
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US10537597B2 (en) 2020-01-21
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